Experience-dependent synaptic plasticity and underlying gene regulation are crucial for normal brain development and learning, and are disrupted in a broad array of disorders of development and learning such as schizophrenia, Alzheimer's disease, drug addiction and age related memory loss. To date, most studies of the cellular and molecular mechanisms of synaptic plasticity and gene regulation have taken a highly reductionist approach in very simplified preparations in vitro. However, practically nothing is known about genomic mechanisms of memory that occur during actual behavioral learning in the intact brain. In particular, the reductionist approach makes it difficult or impossible to study the integration of different inputs and pathways that is critical for many aspects of learning, and is a hallmark of neurodevelopment and associative learning. For these reasons, our long-term objectives are to characterize genome-wide mechanisms of long-term plasticity at the level of single identified neurons during behavioral learning. For this challenging task we will use the well-defined model system of the Aplysia withdrawal reflex, with a nearly complete mapping of a simple memory-forming circuit in a simplified behavioral preparation. We will record the activity of key individually identified neurons in that circuit and the synaptic connections between them during both a nonassociative form of learning (sensitization) and an associative form of learning (classical conditioning). And, for the first time, we will monitor the operation of the entire genome within specific individual neurons as they learn and remember. As a result, we will link neural activity to gene expression, plasticity, and behavior. We will also identify neuron type specific cellular signaling mechanisms and gene regulatory pathways during sensitization and conditioning, and test their roles in long-term plasticity. Based on our previous results, we hypothesize that three signaling pathways (5-HT, NO, and activity) act synergistically to produce more specific and longer-lasting memory traces during conditioning than during sensitization. Furthermore, 5-HT and NO can each change expression at least a thousand genes (some of which overlap) and induce large-scale chromatin remodeling. These findings have raised a fundamental question: how are these different inputs integrated at the level of genome-wide gene regulation in individual neurons in the circuit for conditioning? We will identify critical molecular targets (including promoters, enhancers and relevant transcription factors) leading to such integration, and examine their roles as decision points in the formation of long-lasting memories. These studies will significantly advance our understanding of synaptic and genomic mechanisms that contribute to circuit formation, learning, and memory, and their possible dysfunction in diseases that affect neurodevelopment and memory. PUBLIC HEALTH RELEVANCE: While substantial progress has been made in reductionistic analyses of nonassociative forms of memory in vitro, the molecular and genomic mechanisms of associative forms of memory in vivo are more elusive. We will investigate those mechanisms at all levels (from cell-to-circuit-to-behavior) by directly analyzing molecular and genomic events in individual neurons of the underlying neuronal circuit during behavioral learning. These studies will substantially advance our understanding of normal memory processes and their possible dysfunction in memory related diseases.